Process for treating foods using saccharide esters and superatmospheric hydrostatic pressure

ABSTRACT

In the disclosed treatment of foods or foodstuffs (particularly liquid and semi-liquid or semi-solid foods and solid foods such as meats and stews), the food or foodstuff is subjected to very high hydrostatic pressures in the presence of a saccharide ester (e.g. sucrose mono-, di-, and/or tri-C 8  -C 24  -fatty acid esters) at temperatures above 35° C. but preferably below 60° C. for a relatively short period of time (less than 60 minutes). This treatment is effective in essentially eliminating sporeforming organisms, whether in the form of spores or in the vegetative state.

The U.S. Government has rights in this invention pursuant to ContractNo. DAAK60-95-C-2060 awarded by the Department of the Army.

This application claims benefit of Provisional Appl. 60/065,312 filedNov. 13, 1997.

FIELD OF THE INVENTION

This invention relates to a process for treating foods or foodstuffs forthe purpose of substantially eliminating microorganisms. An aspect ofthis invention relates to a method for elimination of sporulated and/orsporeforming food-spoilage microorganisms from foods. A further aspectof this invention relates to a bactericidal process for treating foodswhich are adversely affected by elevated temperatures but are notsubstantially affected adversely by superatmospheric pressure.

DESCRIPTION OF THE PRIOR ART

A wide variety of microorganisms can be found in raw and partiallyprocessed foods and even in some fully processed foods. Themicroorganisms of greatest concern to food product manufacturers andfood consumers are generally bacteria which produce toxins or which havefood-spoilage effects. Many of these bacteria are temperature-sensitiveand can be killed--or at least significantly reduced in population--byheat treatments such as canning, pasteurization, and aseptic processing.Other means of controlling bacterial populations involve irradiation,hermetically-sealed-in growth-inhibiting atmospheres, and the additionof chemicals such as food preservatives, e.g. sorbates, and/or changesin the natural pH of the foodstuff.

One common practice in this art is to modify the atmosphere in thecontainer which stores the food, thereby providing a safer environmentfor food storage. For example, hermetic or air-tight storage has beenused to seal off the food container and store the food under a gasmixture which contains very little oxygen and a large amount of carbondioxide (compared to the normal atmospheric concentration). The high-CO₂atmosphere in the container prevents growth of oxygen-utilizingorganisms. However, this storage technique does not necessarily hinderthe growth of either anaerobic bacteria or sporeforming microorganisms.

Heat can be a potent weapon against bacteria. Anti-bacterial effects areobtained with thermal processing of the food itself and with thermaltreatment of the containers used to store the food. In thermalprocessing, temperatures can be below 100° C. or heat can be applied inthe form of wet steam (at about 100° C.) or superheated steam (>100°C.). Canning generally involves a steam treatment of the container,whereas processes such as pasteurization (used primarily for dairyproducts and beverages) is a direct treatment of the food itself. Somecontrol over bacterial populations is obtained at temperatures as low as60° C., since the life processes and some essential enzymes are damagedor disrupted and can even become inoperative at temperatures above 37°C. Some enzymes, for example, are completely and permanently denaturedat temperatures as low as 50° C. (By contrast, cooling to temperatureswell below 37°, e.g. 10° C. or less can inhibit or temporarilyinactivate bacterial life processes and enzymes, but the effect is notpermanent, and the bacteria can resume reproductive and other lifeprocesses when the temperature of their environment returns to 20 to 40°C.)

There are, however, serious limitations on the use of heat to killbacteria in foods. Just as enzymes can be denatured by modestly elevatedtemperatures, so can raw and partially processed foods. Even foods whichare normally cooked at relatively high temperatures before beingconsumed oftentimes cannot be sold in a pre-cooked form. It is generallyunacceptable to treat highly temperature-sensitive foods at temperaturesmuch above 60 or even 50° C.

Canning is a severe form of heat treatment that will inactivateheat-resistant microorganisms, including bacterial spores, but due tothe limitations of all heat-treatment methods, described above, canningis most often used for foods such as chunky fruits and soups with meatand vegetables. The canned food of commerce is sterile. Hence, canningis virtually the only food preservation method which is effectiveagainst bacterial spores.

Sporeforming microorganisms which have food-spoilage effects can beparticularly difficult to deal with. Bacterial spores are resistant tomost types of sterilization except for heat treatments in which thetemperature reaches 130 to 145° C. Once these microorganisms have formedspores, the spores tend to be resistant to damage from pressure,moderate temperatures, and many chemical additives.

Sporeforming microorganisms and bacterial spores can resist, inter alia,irradiation treatments and aseptic processing and packaging. Irradiationis useful for protecting raw meats, fruits, dairy products, grains, andvegetables from aerobic, non-sporeforming microorganisms, some molds,and some insects. Bacterial spores are generally unaffected byirradiation.

Aseptic processing/packaging is a sterilization method that wasdeveloped to avoid denaturing foods that cannot withstand the harshconditions of conventional thermal processing. This method usesso-called "UHT" (ultra-high temperature) treatments which are extremelybrief. That is, the heat-sensitive food product is exposed to the UHTtreatment for just a few seconds. Microbial inactivation is achieved,and yet the food product suffers minimal or greatly reduced damage ascompared to conventional heat treatments. The UHT-treated food is thenpackaged in pre-sterilized containers, but the containers have beencooled, so that they do not add to the heat-history of the food product.Unfortunately, as indicated above, UHT sterilization does not killbacterial spores.

Relatively recently, considerable interest has been shown in sugaresters as food preservatives, particularly the mono- and di-saccharidesugars (glucose, fructose, mannose, galactose, sucrose, etc.). Theseesters are typically tasteless, odorless, and non-toxic; moreover theyare essentially non-polluting due to their high degree ofbiodegradability. In addition to food preservation, apparently becauseof bacteriostatic effects, saccharide esters can mimic the effects offat and provide useful emulsifying effects. However, there has as yetbeen no reports in the literature suggesting that saccharide esters canbe bactericidal. Accordingly, in the food preservation field, theseesters appear to be of primary importance for their ability to control,rather than eliminate, bacterial populations.

Accordingly, there is still a serious additional need for treatmentswhich eliminate or virtually eliminate food-spoilage microorganisms,particularly those of the sporeforming type, from temperature-sensitivefoods or foodstuffs.

The following references provide background information onmodified-atmosphere (hermetic sealing), thermal processing, irradiation,and UHT sterilization:

Calderon, M., Food Preservation by Modified Atmospheres, CRC Press,1990, pages 4 to 8;

David, Jairus, Aseptic Processing and Packaging of Food, CRC Press,1996, pages 4 to 19;

Safeguarding the Food Supply Through Irradiation Processing Techniques,International Conference of Agricultural Research Institute, Orlando,Fla., 1992, pages 1 to 28;

Safety and Nutritional Adequacy of Irradiated Food, World HealthOrganization, Geneva, 1994, pages 22 to 28.

SUMMARY OF THE INVENTION

It was now been discovered that a combination of superatmosphericpressure, a saccharide ester, and very moderate temperatures (below therange of cooking temperatures) has major bactericidal effects,particularly upon sporulated or sporeforming microorganisms (e.g.bacilli such as B. subtilis), in treatments lasting a relatively shorttime. This discovery is surprising, since these three factors alone(pressure, saccharide esters, moderate temperatures elevated above roomtemperature, e.g. above about 35° C.), and various combinations of thesethree factors--with the sole exception of all three factorstogether--appear to have negligible, if any, biocidal effects againstthese spores. Although this invention is not bound by any theory, itappears that the three factors mentioned above, given a very modestamount of time, work synergistically to break down spore components andpossibly vegetative envelope components as well. Bactericidal effectsbegin to appear in less than 5 or 10 minutes, and when the treatmentlasts at least 10 minutes, the elimination of sporeforming bacteriaand/or their spores has progressed to a major extent and can be completeor essentially complete. Beyond this modest time requirement, thetreatment time does not appear to be critical, but treatment timeslonger than about an hour are not economical and are hence undesirablefor that reason.

Although superatmospheric hydrostatic pressure, even pressure in thethousands of atmospheres, appears to inflict essentially no damage uponmost types of spores, even at elevated temperatures such as 45° C., andeven though saccharide esters are, apparently, not known to havesignificant wide-range biocidal effects, the high pressures used in theprocess of this invention, at the mildly elevated temperatures utilizedin this invention, appear to cause the saccharide ester to havesporicidal effects vs. bacilli.

The terms "foods" and "foodstuffs" are used substantially synonymouslyin this application, the only difference being that "foods" areconsidered to include edible materials at any stage of processing,whereas "foodstuffs" are more likely to be in raw or partially processedform. Foods and foodstuffs suitable for treatment by this processinclude raw and processed edible (preferably human-edible) materialswhich are acidic (2<pH<7)--preferably mildly acidic, neutral, or verymildly basic (e.g. pH≦about 8) and which are liquid, semi-liquid, orsemi-solid (e.g. foods having a liquid component or which lack rigidityand firm texture such as gravies, jams or jellies, fruit and fruit-baseddeserts, gelatin products and gelatin deserts, salad dressings, dips,salsa and other flavoring mixes, etc.). The liquids, semi-solids, andsemi-liquids can be expected to resist denaturing or other adverseeffects (e.g. upon flavor, color, aroma, or nutrient value) which mightresult from the application of high hydrostatic pressure. Certain solidfoods are also resistant to adverse changes in flavor or nutrient value(and can also resist changes in color and aroma), particularly stews andmeats, and can be treated in accordance with this invention in much thesame manner as semi-solids.

Strongly acid foods (2<pH<5) are of less concern, in the context of thisinvention, as compared to mildly acid and neutral foods.

DETAILED DESCRIPTION

The process of this invention could be considered a form of "coldpasteurization", since the temperatures employed are well below biocidaltemperatures, particularly for spores.

The Saccharide Ester

A key aspect of this invention was the discovery that saccharide esters,normally suitable essentially as bacteriostatic agents, can bebactericidal under the conditions employed in this invention. Althoughthis invention is not bound by any theory, it is presently believed thatthe saccharide ester, in a high hydrostatic pressure environment,interacts with spore layers to disrupt the protective effect of thespore structure and thereby expose the spore to severe damage, e.g. fromthe high pressure. Sporeforming microorganisms, as indicated above, aretypically bacteria, including bacteria of the Bacillus family, e.g. B.subtilis. In foods or foodstuffs treated according to this invention,the sporeforming microorganisms can be present in sporulated and/orvegetative form; the invention is effective against these organismsregardless of their state of sporulation.

The preferred saccharide esters are derived by esterifyingmonosaccharides or disaccharides, most preferably disaccharides, such assucrose, C₁₂ H₂₂ O₁₁, which is made up of one hexose unit linked to afuranose unit: ##STR1##

An important structural feature of the sucrose molecule is its threemethylol groups (--CH₂ OH), all of which are sterically unhindered andare easier to esterify with carboxylic acid esterifying agents (e.g.carboxylic acids, carboxylic acid halides, and carboxylic acidanhydrides) than are the five ring-substituted hydroxyl groups. Thus,although sucrose can be esterified completely to the octa-ester, themono-ester tends to form first, followed by the di- and tri-esters. Thesaccharide esters of this invention are preferably mono-, di-, and/ortri-esters, and it is further preferred that the mono-ester content ofthe esterified product be at least 10% by weight of the total product,essentially the balance being di- and tri-esters. In a particularlypreferred embodiment of this invention, the major amount by weight ofthe esterified disaccharide is the mono-ester, and a mono-ester contentas high as about 80 or 90% by weight is particularly useful. It is notnecessary, however, to eliminate all of the higher ester content, nor isit practical. Thus, the total amount of di- and/or tri-ester content isgenerally not less than about 10% by weight.

Generally speaking, the hydrophile/lipophile balance (HLB) of saccharideesters is affected by the number of hydroxyl groups esterified, and thehigher esters (particularly sucrose octa-esters) can be extremelylipophilic--probably too lipophilic to have sufficient biocompatibilitywith the target for attack. It presently appears that a certain amountof both hydrophilic and lipophilic properties are desirable for thepurpose of breaking down the integrity of the spore, and the HLB ofdisaccharide mono-esters, di-esters, and tri-esters appears to be inapproximately the correct range for achieving the objectives of thisinvention.

Another factor which affects biocompatibility and the HLB range is thenature of the carboxylic acid residue of the ester groups.Straight-chain fatty acids are preferred, and the HLB is considered toolow in lipophilic properties if the carbon chain of the fatty residuehas less than about 8 carbons. The hydrocarbon character of fatty acidshaving a chain longer than about 24 carbons, on the other hand, isbelieved to be excessive. The peak values for good HLB balance andcompatibility with spore components are believed to lie in the range offatty acid residues having about 10 to about 20 carbon atoms. Althoughboth saturated and unsaturated straight-chain fatty acid residues aresuitable in the context of this invention, the preferred residues aresaturated (e.g. lauric, palmitic, myristic, and stearic acids).

Especially good results have been obtained with a sucrose lauratecontaining about 70 to 80% of the monoester, essentially the balancebeing di- and/or tri-ester. The preferred esterification site is themethylol group of the hexose unit of the sucrose molecule.

Thus, a preferred embodiment of sucrose ester is represented by formulaI ##STR2## where R¹ is an alkyl group having from 10 to 20 (especially12 to 18) carbon atoms, and R² and R³ are H or, less preferably, C₁₀-C₂₀ -alkyl.

The most effective way of carrying out the process of this invention inthe presence of the saccharide ester is to add the saccharide ester tothe food or foodstuff, prior to processing, in generally theconventional ways that food additives are introduced. For example in thecase of liquid and semi-liquid foods, the additive can be thoroughlyblended with the food using a mixer. Since the saccharide esters areavailable in powder and waxy forms, solid foods such as meats can begiven a very complete surface coating of the ester.

Typical amounts of saccharide ester added to the food or foodstuff priorto processing range from about 0.1 parts by weight per 100 parts byweight of product (0.1 phr) to about 1.0 phr.

Processing Conditions

As noted previously, foods and foodstuffs selected for treatmentaccording to this invention are generally not harmed by high hydrostaticpressure. However, it is well known in the food art that some changes inflavor, aroma, and color can occur during food processing withoutrendering the food unsuitable for sale and consumption. The most seriousadverse processing changes are considered to be major loss of nutrientvalue and the creation of unacceptable flavors.

To impart bactericidal properties to the saccharide ester, thesuperatmospheric hydrostatic pressure should be very high--in thethousands of atmospheres, e.g above 3,000 atmospheres (above about 310MPa). Optimum results have been obtained with about 4,000 atmospheres(about 410 MPa). Still higher pressures can be used, but do not appearto provide any additional advantages.

Although this invention is not bound by any theory, it is believed thatthe function of the treatment temperature has less to do with outrightkilling of bacteria than it does with facilitating the interactionbetween the saccharide ester and the spore cortex. Accordingly, it isnot necessary that the temperature at which the process is conducted besignificantly above 35 or even 37° C. Even a temperature of only 40° C.will, apparently, facilitate this interaction. The optimum temperatureappears to be as low as about 40 to 50° C. Higher temperatures can beused (e.g 60° or more), but they do not appear to confer any significantadvantage, and they increase the risk of lowering sensory quality orreducing the nutrient value of the product. Accordingly, this inventionappears to be ideally suited to the treatment of foodstuffs which arenot adversely affected by pressure but which are subject to heatdenaturization.

The effectiveness of temperatures utilized in this invention is believedto be surprising, since, in the absence of the high hydrostatic pressureand the saccharide ester, thermal treatments in the 100 to 250° C. rangeare normally required to have a significant impact upon a sporepopulation. As noted previously, there are many foods and foodstuffswhich cannot be heated to such high temperatures without detractingseriously from their commercial value.

Treatment times have been discussed previously. Generally speaking, thetreatment (especially the pressure, which is the most difficult tomaintain) can be continued until the spore integrity have beensubstantially totally disrupted or broken down. Microorganisms in thevegetative state are also essentially destroyed. Further continuation ofthe treatment beyond this point adds cost and complexity withoutsignificantly improving the product of the process. For example,treatment times longer than 60 minutes appear to serve no importantpurpose and are wasteful of energy and equipment time. Essentialelimination of the sporeforming organisms, whether in spore form or inthe vegetative state, has been observed with treatment times as short asabout ten minutes. Where less complete elimination of thesemicroorganisms can be tolerated, times shorter than 10 minutes can beused, but apparently, very little is accomplished in less than about 2minutes. Treatment times of at least 5 minutes are preferred.

The design of continuous processing equipment which provides a highlypressurized zone is complex, hence batch or semi-continuous processingis preferred for use in this invention. The application of very highhydrostatic pressures in a sealed, pressurized chamber or zone can bereadily provided with commercially available batch-processing equipment,and the temperatures required by this invention pose no additionaldifficulties.

Post-Treatment Considerations

When the high-pressure treatment of this invention is completed, thepressure can be released, e.g. to normal ambient temperature, byunsealing or depressurizing the pressurized chamber. The temperature ofthe resulting treated product can be maintained at 35° C. or higher fora short time after treatment, but it is often most desirable torefrigerate the product after pressure treatment. Cooling of the treatedproduct to refrigeration or freezing temperatures below 15° C. (e.g. -20to 10° C.) is generally preferred. Deep freezing to temperatures as lowas -60° C. can also be used. The product is thus preferably kept underrefrigeration for storage or shipping.

EXAMPLE

Microbiological media representing model foods and containing asignificant population of bacterial spores were blended with 0.1% ofsucrose laurate type L-1695 (Ryoto Sugar Ester, a product of MitsubishiChemical Corporation). The L-1695 sucrose laurate is a waxy powderhaving an HLB value of 16 and contains 80% of the mono-laurate (theesterification being on the methylol group of the hexose unit of thesucrose). Essentially the balance of the Ryoto product is believed to besucrose di-laurate and/or sucrose tri-laurate. The medium containing thesucrose laurate was placed in a mildly heated pressure unit andsubjected to 4,000 atmospheres (410 MPa) for ten minutes at 45° C. Thepressure was released, and the samples are removed from the pressureunit. Substantially complete elimination of the spore population wasobserved.

What is claimed is:
 1. A process for treating food, comprising the stepof:subjecting the food to a superatmospheric pressure which hasbactericidal effects in the presence of a saccharide monocarboxylic acidester which comprises at least 10% by weight of a mono-ester of theformula I ##STR3## wherein R is an alkyl group having from 10 to 20carbon atoms, essentially the balance of said monocarboxylic acid esterbeing a corresponding diester or triester or mixture thereof and at atemperature above room temperature but below the temperature at whichthe food will be adversely affected in flavor or nutrient content byheat denaturization.
 2. The process according to claim 1, wherein thefood contains sporulated or sporeforming microorganisms or a mixturethereof, wherein said temperature is above about 37° C., whereinsaccharide monocarboxylic acid ester comprises a monoester of a C₈ - toC₂₄ -carboxylic acid, and wherein said superatmospheric pressure andsaid temperature are maintained until the sporulated microorganisms andsporeforming microorganisms or mixtures of sporeforming and sporulatedmicroorganisms in said food are essentially eliminated.
 3. The processaccording to claim 2, wherein the saccharide of said monocarboxylic acidester is a disaccharide.
 4. The process as claimed in claim 1, whereinsaid food is liquid, semi-liquid, semi-solid having a liquid componentor which lacks rigidity.
 5. The process as claimed in claim 4, whereinsaid food is gravy, jam, jelly, fruit, fruit-based desert, a gelatinproduct, a salad dressing, a dip, salsa, flavoring mix, stew or meat. 6.A process for essentially eliminating, from a food, at least onepopulation of sporeforming microorganisms, whether in sporulated and/orvegetative form, said process comprising:a. mixing a liquid,semi-liquid, or semi-solid food which essentially lacks the rigidity ofa solid food and which contains at least one population of said sporeforming microorganisms with a saccharide ester composition, saidsaccharide ester composition comprising at least 10% by weight of amono-ester of the formula I ##STR4## wherein R is an alkyl group havingfrom 10 to 20 carbon atoms, essentially the balance of said saccharideester being a corresponding diester or triester or mixture thereof, b.subjecting the food containing said saccharide ester composition tosuperatmospheric hydrostatic pressure and to a temperature above roomtemperature but below the temperature at which the food will beadversely affected in flavor or nutrient content by heat denaturization,said superatmospheric hydrostatic pressure and said temperature beingmaintained until said population of sporeforming microorganisms, whetherin sporulated or vegetative form, has been essentially eliminated, c.releasing said superatmospheric hydrostatic pressure, and d. recoveringthe resulting treated food which is essentially free of sporeformingmicroorganisms, whether in sporulated or vegetative form.
 7. The processaccording to claim 6, wherein said superatmospheric hydrostatic pressureis approximately 4,000 atmospheres, and said temperature ranges fromabout 40 to about 50° C.
 8. The process according to claim 7, whereinthe saccharide of said saccharide monocarboxylic acid ester is adisaccharide.
 9. The process as claimed in claim 8, wherein saidcompound of formula I consists essentially of sucrose monolaurate, andwherein said disaccharide monocarboxylic acid ester comprises a majoramount of said compound of formula I.
 10. The process as claimed inclaim 6, wherein said saccharide monocarboxylic acid ester comprises atleast 10% by weight of a C₁₀ -C₂₀ -carboxylic acid mono-ester ofsucrose, essentially the balance of said saccharide monocarboxylic acidester being a corresponding di-ester or tri-ester or mixture thereof.11. The process according to claim 6, comprising the steps of:a. addingsaid saccharide monocarboxylic acid ester to said food, b. subjectingthe food containing said saccharide monocarboxylic acid ester to saidsuperatmospheric hydrostatic pressure at a temperature in the range ofabout 40 to about 50° C., and c. releasing said superatmospherichydrostatic pressure after a period of time exceeding 5 minutes but notexceeding about 60 minutes.
 12. The process according to claim 11,wherein said saccharide monocarboxylic acid ester is a sucrose mono-,di-, or tri-ester, or a mixture thereof, and the monocarboxylic acidresidue of said monocarboxylic acid ester is a fatty acid having fromabout 10 to about 20 carbon atoms.
 13. A process according to claim 6,wherein said saccharide ester composition contains a minor amount ofdi-ester, a minor amount of a tri-ester, or a mixture thereof, saiddi-ester and tri-ester being C₈ -C₂₄ -carboxylate esters.
 14. A processaccording to claim 6, wherein, in said step b, said superatmospherichydrostatic pressure and said temperature are maintained for a period oftime exceeding five minutes but not exceeding about 60 minutes.
 15. Aprocess according to claim 6, wherein said food is capable ofwithstanding superatmospheric pressure in excess of 3,000 atmosphereswithout being adversely affected in flavor or nutrient value, andwherein said superatmospheric hydrostatic pressure applied during saidstep b is greater than 3,000 atmospheres.
 16. A process according toclaim 6, wherein said temperature is greater than about 37° C.
 17. Aprocess according to claim 16, wherein said temperature is greater thanabout 40 but less than about 60° C.
 18. A process according to claim 6,wherein, in said step a, the food is mixed with sucrose monoester, saidsucrose monoester optionally containing sucrose di-ester and/ortri-ester, said sucrose monoester, di-ester, and tri-ester being C₁₀-C₂₀ -carboxylates.
 19. The process according to claim 6, wherein saidcompound of formula I consists essentially of sucrose monolaurate.
 20. Aprocess according to claim 6, wherein the minor amount of saidsaccharide ester composition is about 0.1 to about 1 part of saidcomposition per 100 parts of said food.
 21. The process according toclaim 6, comprising the additional steps of decreasing saidsuperatmospheric hydrostatic pressure to approximately normal ambientpressure, cooling the resulting treated food to a refrigerationtemperature below about 15° C., and maintaining the resulting treatedfood at the refrigeration temperature for storing or shipping.
 22. Aprocess for essentially eliminating, from a solid food, at least onepopulation of sporeforming microorganisms. whether in sporulated and/orvegetative form, said process comprising:a. coating the surface of asolid food which contains at least one population of said sporeformingmicroorganisms with a powdered or waxy sucrose-C₁₀ -C₂₀ -carboxylate, b.subjecting the food thus coated with said saccharide ester compositionand wherein said saccharide ester composition comprises at least 10% byweight of a monoester of the formula I ##STR5## wherein R is an alkylgroup having from 10 to 20 carbon atoms, essentially the balance of saidsaccharide monocarboxylic acid ester being a corresponding diester ortriester or mixture thereof,to superatmospheric hydrostatic pressure andto a temperature above room temperature but below the temperature atwhich the food will be adversely affected in flavor or nutrient contentby heat denaturization, said superatmospheric hydrostatic pressure andsaid temperature being maintained until said population of sporeformingmicroorganisms, whether in sporulated or vegetative form, has beenessentially eliminated, c. releasing said superatmospheric hydrostaticpressure, and d. recovering the resulting treated food which isessentially free of sporeforming microorganisms, whether in sporulatedor vegetative form.